Gene Mining and Expression of α-glucosyltransferase from Lactobacillus plantarum
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Abstract:
The α-glucosyltransferase (α-GTF) is a type of multifunctional enzyme that can synthesize oligosaccharides using maltose, isomaltose, and O-methyl glucoside as substrates, and has attracted attention because of its functional diversity and specificity. In this study, the whole genome information of Lactobacillus plantarum (FMNP01), which was sequenced by our team, and the bioinformatics approach were used to analyze the α-gtf gene obtained from Lactobacillus. Meanwhile, the primers were designed based on the DNA sequences of the α-gtf gene in Lactobacillus, which are listed in the National Center for Biotechnology Information (NCBI) database. Three α-gtf genes were cloned by polymerase chain reaction (PCR) from Lactobacillus plantarum, Lactobacillus casei, and Lactobacillus brevis, and the similarities of the encoded amino acid sequences were 42%, 29%, and 29%, respectively. The three obtained genes were cloned into vector pET-32a to construct the recombinant pET-32a-α-gtf, which were then transformed into the expression host Escherichia coli BL21 to induce expression. The fusion proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the results showed that there was an obvious band at 75 ku, consistent with the molecular weight of the predicted protein. The results indicated that three α-gtf genes were expressed successfully. Meanwhile, the inducer concentration and induction time and temperature were optimized, and this study is expected to lay the foundation for related studies in the future.
